Novel MLCK-MIF Interaction in Endothelium

内皮细胞形成脉管系统和许多组织之间的界面，并且是渗透性、炎症、新血管形成和凝血的主要参与者。我们的实验室已经确定了内皮细胞骨架在血管病理生物学中的关键作用。然而，尽管其公认的重要性，在这些过程中的细胞骨架调节的确切机制还不完全清楚。我们的工作已经证明了由Ca+2 - CaM依赖的内皮细胞肌球蛋白轻链激酶（EC MLCK）催化的肌球蛋白轻链（MLC）磷酸化在血管通透性、中性粒细胞跨内皮迁移、细胞运动和凋亡中的关键作用。我们克隆了这种酶，发现EC MLCK含有平滑肌MLCK中不存在的独特氨基末端序列。使用酵母双杂交系统，发现EC MLCK的N-末端部分与促炎细胞因子巨噬细胞迁移抑制因子（MIF）相互作用，MIF是先天性和获得性免疫的调节因子，在ARDS、脓毒症和关节炎中具有病理作用。事实上，抗MIF抗体在海洋内毒素血症模型中具有保护作用。使用GST-MLCK下拉和免疫沉淀试验，我们已经证实了这种高度新颖的EC MLCK和MIF之间的相互作用的特异性。然而，这种相互作用的生物学意义尚不清楚。在特定目标#1中，MLCK和MIF将在人肺动脉内皮细胞内空间共定位。具体目标#2将使用体外结合试验表征EC MLCK和MIF的新N-末端内相互作用所必需的结构位点。最后，在具体目标#3中，将评价MLCK-MIF相互作用在渗透性、中性粒细胞跨内皮迁移、内皮细胞趋化性和细胞凋亡中的生物学意义。这些研究将共同确定多功能MLCK和高度相关的细胞因子MIF在关键内皮病理过程中的新型相互作用的贡献。

Endothelial cells form the interface between the vasculature and many tissues and are major participants in permeability, inflammation, neovascularization, and coagulation. Our laboratory has defined a critical role for the endothelial cytoskeleton in vascular pathobiology. However, despite its recognized importance, the exact mechanisms operative is cytoskeletal regulation in these processes are incompletely understood. Our work has demonstrated a critical role of phosphorylation of myosin light chains (MLC) catalyzed by the Ca+2 - CaM dependent endothelial cell myosin light chain kinase (EC MLCK) in vascular permeability, neutrophil transendothelial migration, cell motility, and apoptosis. We have cloned this enzyme and found that EC MLCK to contains an unique amino terminal sequence not present in smooth muscle MLCK. Using the yeast two-hybrid system, the N-terminal portion of EC MLCK was found to interact with a proinflammatory cytokine, macrophage migration inhibitory factor (MIF), a regulator of innate and acquired immunity with a pathologic role in ARDS, sepsis, and arthritis. Indeed, anti-MIF antibodies are protective in a marine model of endotoxemia. Using GST-MLCK pull-down and immunoprecipitation assays, we have confirmed the specificity of this highly novel interaction between EC MLCK and MIF. However, the biological significance of this interaction is not known. In Specific Aim #1, MLCK and MIF will be spatially colocalized within human pulmonary artery endothelial cells. Specific Aim #2 will characterize the structural sites within both the novel N- terminus of EC MLCK and MIF necessary for interaction using in vitro binding assays. Finally, in Specific Aim #3 the biologic significance of MLCK-MIF interaction in permeability, neutrophil transendothelial migration, endothelial cell chemotaxis, and apoptosis will be evaluated. Together these studies will define the contribution of the novel interaction between the multifunctional MLCK and a highly relevant cytokine MIF in key endothelial pathologic processes.